1. Field of the Invention
The present invention relates to an enzyme-labeled immunoassay comprising the steps of allowing a test sample to react with an enzyme-labeled reagent, allowing a substrate to react with the enzyme to form a signal, and immobilising the enzyme-labeled reagent, with the prevention of a further signal formation from a pre- determined time on after the immobilisation of the enzyme-labeled reagent, using an enzyme inhibitor. The present invention also relates to a device for performing this enzyme-labeled immunoassay.
2. Discussion of Background
Recently immunoassays using an enzyme as a labeling material and a device comprising an absorbent material capable of transporting an enzyme-labeled reagent by capillary action, using a developing solution, are known as convenient and simple assays for the detection of analyte substances in biological fluids, and medicinal substances in test samples, utilizing immunoreactions (refer to, for example, Japanese Laid-Open Patent Application 61-145459, European Patent 186,799, Japanese Laid-Open Patent Application 63-501595, U.S. Patent 5,604,110 and European Patent 225,054). When using a test strip comprising such a device, for example, a test sample containing an analyte is allowed to react with an enzyme-labeled reagent, a substrate is then allowed to react with the enzyme to form a signal, and immobilising the enzyme-labeled reagent reacted with the test sample, using an immunoreactive substance in an indicator reagent zone, and in a predetermined period of time after the reaction of the test sample and the enzyme-labeled reagent, the degree of the formation of the signal, such as the coloring degree of the indicator reagent zone, is measured or evaluated, whereby the amount of the analyte contained in the test sample is assayed.
In order to prevent the formation of the signal (coloring or luminescence) except where enzyme-labeled reagent is immobilized at the indicator reagent zone, there is proposed an immunoassay test strip in which a signal formation inhibitor is contained therein to prevent the reaction between the enzyme and the substrate until the developing solution reaches the indicator reagent zone (Japanese Laid-Open Patent Application 1-503439, U.S. Patent 5,641,639, European Patent 312,565, Japanese Laid-Open Application 5-149951 and European Patent 512,390). In such an immunoassay using the above test strip, it is possible to prevent the formation of the signal up to the indicator reagent zone by providing a signal formation inhibitor zone upstream of the indicator reagent zone in terms of the transport direction of the developing solution, optionally with the provision of a signal initiation zone, whereby immunoassay can be carried out with high sensitivity for many analysis items.
However, in the immunoassay using the above-mentioned signal formation inhibitor, it is possible to inhibit the formation of the signal by the reaction between the enzyme and the substrate from the initiation of the assay with the application of the developing solution to the strip until the developing solution reaches the indicator reagent zone, but cannot inhibit the formation of the signal which continuously take place in the indicator reagent zone after a specific period of time.
Further, in the immunoassay using a coloring substrate, the degree of the coloring of the indicator reagent zone is evaluated by visual inspection or using a color difference meter after the reaction between the enzyme and the substrate for a predetermined period of time, whereby an assay result is obtained. In such a conventional immunoassay, when a negative or positive evaluation or judgement is performed, using the difference in the density of the coloring in the indicator reagent zone, the density of the coloring increases with time in the course of the assay, so that the evaluation or judgement may be changed from "negative" to "positive." Therefore, it is necessary to perform the assay with the assay time thereof being strictly controlled when the assay is performed, evaluating the changes in the density of the coloring in the indicator reagent zone.
Under such circumstances, there is demanded for an enzyme-labeled immunoassay which is capable of providing an accurate assay result, with the formation of a signal in the indicator reagent zone which depends upon the assay result, but without further signal formation from a predetermined time on in the course of the assay.